Effect of carrier oil on α-TOCOPHEROL encapsulation in ora-pro-nobis (Pereskia PDF

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Effect of carrier oil on α-TOCOPHEROL encapsulation in ora-pro-nobis (Pereskia

aculeata Miller) mucilage-whey protein isolate microparticles
Isabelle Cristina Oliveira Neves, Sérgio Henrique Silva, Natália Leite Oliveira,
Amanda Maria Teixeira Lago, Natalie Ng, Arianna Sultani, Pedro Henrique Campelo,
Lizzy Ayra Alcântara Veríssimo, Jaime Vilela de Resende, Michael A. Rogers
PII: S0268-005X(19)32472-5
DOI: https://doi.org/10.1016/j.foodhyd.2020.105716
Reference: FOOHYD 105716
To appear in: Food Hydrocolloids
Received Date: 21 October 2019

Revised Date: 11 January 2020
Accepted Date: 26 January 2020
Please cite this article as: Neves, I.C.O., Silva, Sé.Henrique., Oliveira, Natá.Leite., Lago, A.M.T., Ng, N.,
Sultani, A., Campelo, P.H., Veríssimo, Lizzy.Ayra.Alcâ., de Resende, J.V., Rogers, M.A., Effect of carrier
oil on α-TOCOPHEROL encapsulation in ora-pro-nobis (Pereskia aculeata Miller) mucilage-whey protein
isolate microparticles, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105716.
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Credit author statement
Isabelle Cristina Oliveira Neves: Project administration, Conceptualization, Methodology,

Investigation, Writing – Original Draft. Sérgio Henrique Silva: Conceptualization, Methodology.
Natália Leite Oliveira: Formal analysis, methodology. Amanda Maria Teixeira Lago: Formal
analysis, Visualization. Natalie Ng: Investigation, Conceptualization. Arianna Sultani:
Investigation, Data Curation. Pedro Henrique Campelo: Methodology, Conceptualization,
Review. Lizzy Ayra Alcântara Veríssimo: Conceptualization, Methodology. Jaime Vilela de
Resende: Project administration, Resources, Supervision, Funding acquisition. Michael A.
Rogers: Project administration, Resources, Methodology, Formal analysis, Supervision, Writing-
Review and Editing.
1
1 EFFECT OF CARRIER OIL ON α-TOCOPHEROL ENCAPSULATION IN

2 ORA-PRO-NOBIS (Pereskia aculeata Miller) MUCILAGE-WHEY PROTEIN
3 ISOLATE MICROPARTICLES
4
5 Isabelle Cristina Oliveira Nevesa,b, Sérgio Henrique Silvaa, Natália Leite Oliveiraa,
6 Amanda Maria Teixeira Lagoa, Natalie Ngb, Arianna Sultanib, Pedro Henrique
7 Campeloc, Lizzy Ayra Alcântara Veríssimoa, Jaime Vilela de Resendea, Michael A.
8 Rogersb*
9
a
10 Department of Food Science, Federal University of Lavras, Lavras, MG, 37200-900,
11 Brazil.
b
12 Department of Food Science, University of Guelph, Guelph, ON N1G2W1, Canada.
c
13 Faculty of Agrarian Science, Federal University of Amazonas, Manaus, AM,
14 69077-000, Brazil.
15
16 *Corresponding author:
17 M.A. Rogers
18 E-mail address: mroger09@uoguelph.ca
19 TEL. +01 519 924-4120 EXT. 54327
2
20 ABSTRACT
21 Microparticles of whey protein isolate (WPI) and ora-pro-nobis mucilage (OPN)
22 encapsulated α-tocopherol were made using long-chain unsaturated (e.g. canola oil
23 (CA)) or medium-chain saturated oil (e.g., coconut oil (CO)) as the carrier oil.
24 Microparticles were produced from CO- or CA-in-water emulsions by freeze-drying
25 emulsions with various ratios of WPI/OPN. Before freeze dying, emulsions exhibited
26 Newtonian or shear-thinning behavior. Drying yields for freeze-dried emulsions ranged
27 between 74.1% to 87.1% w/w, depending on the biopolymers-to-oil ratio and varied
28 depending on whether CA or CO was used as the carrier. WPI:OPN ratios (between
29 23:1 and 7:1) nor oil phase (e.g. CO or CA) significantly affected the physical
30 properties (e.g., oil retention, water content, and activity) of the dried powder between
31 treatments. Higher powder bulk density (0.22 g·cm-3) and encapsulation efficiency
32 (79.8% w/w) were obtained from freeze-drying CO-, compared to CA-in-water
33 emulsions and with higher concentrations of OPN. Over 35 days, α-tocopherol retention
34 and degradation kinetics differed between CO and CA and was dependent on relative
35 humidity. Bioaccessibility of encapsulated α-tocopherol was higher with WPI/OPN and
36 CA (55.0 ± 1.89%) compared to CO (42.4 ± 1.78%), while the rate of α-tocopherol
37 release and induction time for release were statically equal.
38
39 Keywords: Vitamin E; Canola oil; Coconut oil; Degradation kinetics; Isothermal
40 behavior; Carrier oil; Bioaccessibility.
41
3
42 1. Introduction
43 Bioactive encapsulation in foods, cosmetics, and pharmaceuticals improves
44 bioavailability and stability and as such has experienced a recent surge in interest
45 (Koshani & Jafari, 2019; Rafiee, Nejatian, Daeihamed, & Jafari, 2019; Taheri & Jafari,
46 2019). Encapsulation protects bioactives from harsh storage conditions (i.e., exposure to
47 oxygen, moisture, temperature, & light), inhospitable luminal conditions (i.e., presence
48 of enzymes & acidic pH) while masking undesirable flavors and odors (Ballesteros,
49 Ramirez, Orrego, Teixeira, & Mussatto, 2017). Improving bioavailability, especially for
50 essential fat-soluble bioactives, is an active area of investigation due to their poor
51 aqueous solubility in the luminal track hindering them from performing their biological
52 function due to notoriously low bioaccessibility, the precursor to bioavailability (Amiri
53 et al., 2018). Bioaccessibility refers to the amount of bioactive released from the food or
54 encapsulates into the gastrointestinal (GI) luminal fluid that is then available to be
55 transported across the epithelial layer of the GI tract. In vitro bioaccessibility is most
56 accurately approximated by dynamically simulating the luminal conditions (i.e.,
57 peristaltic contractions, continuous gastric and intestinal emptying, pH, enzyme
58 excretion and metabolite absorption) (AlHasawi et al., 2017; Bandali, Wang, Lan,
59 Rogers, & Shapses, 2018; M. Minekus, 2015; Mans Minekus, Marteau, Havenaar, &
60 Huisintveld, 1995; Ting, Zhao, Xia, & Huang, 2015).
61 Encapsulation of hydrophobic and amphiphilic bioactives commonly utilizes
62 emulsions, either as a precursor or final delivery vehicle, as they facilitate incorporation
63 of hydrophobic regions of bioactives into the lipophilic dispersed phase and the
64 hydrophilic region resides in the aqueous phase. Bioactive incorporation into emulsions
65 improves loading efficiency and slows deteriorative reactions; thereby increasing both
66 processing and storage stability (Carneiro, Tonon, Grosso, & Hubinger, 2013;
67 Fioramonti, Rubiolo, & Santiago, 2017). Of particular interest herein, the role of carrier
68 oil (CO or CA) on microparticle properties and bioaccessibility examines the effects of
69 unsaturated versus saturated oil on bioactive stability and release in OPN. Canola oil is
70 comprised primarily of long-chain, unsaturated fatty acids (Madankar, Dalai, & Naik,
71 2013), while coconut oil is high in medium-chain, saturated fatty acids (Beneš, Paruzel,
72 Trhlíková, & Paruzel, 2017; Costa et al., 2016). These were selected as the lipid carrier
73 due to the drastic difference in chemical composition which potentially alters
74 encapsulation efficiency and bioaccessibility. Additional processing is required to
75 convert oil-in-water loaded emulsions into microparticles.
4
76 Microencapsulation forms a protective boundary layer around bioactives which

77 may be engineered to release bioactives via external triggers, such as pH, that control
78 site specific release and disintegration (i.e., release) kinetics thereby reducing
79 detrimental reactions capable of altering bioactive structure and thus eliminating
80 biological activity (Tonon, Grosso, & Hubinger, 2011; Tyagi, Kaushik, Tyagi, &
81 Akiyama, 2011). Numerous drying methodologies produce microparticles, however, for
82 thermally labile bioactives, such as vitamin E (Vit E), freeze-drying is advantageous
83 because of the low operating temperatures and pressures that slow oxidative
84 deterioration (Arab et al., 2019; Ballesteros et al., 2017; da Cruz, Dagostin, Perussello,
85 & Masson, 2019; da Rosa et al., 2013; Ezhilarasi, Karthik, Chhanwal, &
86 Anandharamakrishnan, 2013; Gursul, Karabulut, & Durmaz, 2019; Khazaei, Jafari,
87 Ghorbani, & Kakhki, 2014; Laokuldilok & Kanha, 2015; Martin, de Freitas, Sassaki,
88 Evangelista, & Sierakowski, 2017; Ozkan, Franco, De Marco, Xiao, & Capanoglu,
89 2019; Pasrija, Ezhilarasi, Indrani, & Anandharamakrishnan, 2015; Sanchez, Baeza, &
90 Chirife, 2015; Shaddel et al., 2018; Yamashita et al., 2017). α-tocopherol, the most
91 biologically active form of Vit E, mitigates risks associated with cardiovascular disease,
92 minimizes the effects of aging and reduces the likelihood of diseases initiated by free
93 radicals (i.e., inflammation & cancers) (Reboul, 2017; Sharif et al., 2017).
94 Unfortunately, throughout Western society, ultra-processed foods are rapidly displacing
95 whole foods as the staple food (Reboul, 2017; Traber, 2014; Troesch, Hoeft, McBurney,
96 Eggersdorfer, & Weber, 2012); and this change is in part responsible for consumers
97 intaking less than the Recommended Dietary Allowance (RDA) for Vit E (EFSA Panel
98 on Dietetic Products Nutrition and Allergies, 2015; Institute of Medicine, 2000). Further
99 complicating the problem, instability, and insolubility of α-tocopherol lead to lower than
100 expected bioavailability (Ye, Astete, & Sabliov, 2017). Research on storage stability
101 and digestion kinetics of encapsulated Vit E is critical to actualizing its benefits.
102 No one-fit solution exists to design microparticles as they can be engineered
103 using lipids, proteins, surfactants, polysaccharides or any combination thereof.
104 Although microencapsulation is possible using a single polymer, multi-component
105 encapsulation is generally preferred because each polymer elicits different properties to
106 the encapsulate. Blending polymers provides an exciting opportunity to more precisely
107 engineer the final properties of the protective layers (Do, Hadji-Minaglou, Antoniotti, &
108 Fernandez, 2015). The selection of the microparticle wall material depends on the
109 encapsulating technique, physical properties of the core material, and cost (Alvarenga
5
110 Botrel et al., 2012; Korma et al., 2019; Scholten, Moschakis, & Biliaderis, 2014). Raw
111 materials derived from nature, such as polysaccharides and proteins, are preferred by
112 consumers, as they are generally recognized as safe (GRAS), affordable and
113 biodegradable (Rehman et al., 2019). Research and development of raw materials with
114 ‘clean’ ingredient labels are sought-after by industry to completely or partially replace
115 synthetic ingredients applied to foods and beverages (Adjonu, Doran, Torley, &
116 Agboola, 2014; Ozturk & McClements, 2016; Yang & McClements, 2013).
117 Whey proteins present several desirable functional properties, such as
118 emulsifying capacity, gelation and film-forming ability, as well as active surface
119 properties (Kelly, 2019; Suhag & Nanda, 2015). Recent studies have shown utility of
120 using whey proteins to encapsulate and controlled release of bioactives, because it
121 forms a microparticle wall surrounding the lipid core, either as a single component or in
122 combination with others, presenting high encapsulation yield and storage stability
123 (Bilek, Yılmaz, & Özkan, 2017; Devi, Sarmah, Khatun, & Maji, 2017; Esfanjani, Jafari,
124 & Assadpour, 2017; Shishir, Xie, Sun, Zheng, & Chen, 2018; Tan, Zhong, & Langrish,
125 2020). Recently, the emulsifying and stabilizing activity of Pereskia aculeata Miller,
126 popularly known as ora-pro-nobis (OPN) mucilage has been investigated (Martin et al.,
127 2017) for gel-forming capacity and emulsions stability (Conceição, Junqueira, Guedes
128 Silva, Prado, & De Resende, 2014; Junqueira, Amaral, Oliveira, Prado, & de Resende,
129 2018; Lima Junior et al., 2013). Alone, OPN produces stable ultrasound-assisted
130 nanoemulsions (Lago et al., 2019); but, no studies exploring the effects of carrier oil on
131 OPN mucilage/whey protein isolate (WPI) blends on the microencapsulation of
132 bioactive compounds have been reported to date. OPN is a non-toxic mucilage extracted
133 from the Cactaceae family (Martin et al., 2017). OPN leaves have high concentrations
134 of proteins attached to arabinogalactan chains composed by galactose, arabinose,
135 rhaminose and galacturonic acid (Lima Junior et al., 2013; Martin et al., 2017). This
136 work aims to characterize the physical properties, storage stability, and bioaccessibility
137 of OPN mucilage/WPI microparticles containing α-tocopherol dispersed in CA or CO
138 freeze-dryied oil-in-water emulsions.
139
140 2. Materials and methods
141 2.1. Materials
142 Ethyl alcohol (95%), methanol (≧ 99.9%), hexane (≧ 95%), hydrochloric acid
143 (37%), sodium hydroxide (97%, flakes), α-tocopherol (synthetic, ≧ 96%), sodium
6
144 bicarbonate (≧ 99.7%), (hydroxypropyl)methylcellulose (HPMC, mol. Wt. ~86 kDa),

145 bile extract porcine ((95% (50% cholic acid sodium salt and 50% deoxycholic acid
146 sodium salt)), sodium chloride anhydrous (≧ 99%), potassium chloride anhydrous (≧
147 99%), calcium chloride dihydrate (≧ 99%), lipase from porcine pancreas (type II, 30-90
148 units/mg protein using triacetin), pepsin from porcine gastric (≧ 2,500 units/mg
149 protein), α-amylase from Bacillus sp. (type II-A, ≧ 1,500 units/mg protein using biuret),
150 pancreatin from porcine pancreas (4 x USP), trypsin from bovine pancreas (≧ 7,500
151 BAEE units/mg solid) were obtained from Sigma Aldrich (MO, USA). Acetonitrile (≧
152 99.9%) and petroleum ether (HPLC and GC grade) were purchased from Fisher
153 Scientific (NJ, USA). Canola oil (Great Value, ON, CA) and coconut oil (Great Value,
154 ON, CA) were acquired from local supermarkets. Whey protein isolates Lacprodan®
155 DI-9213 was kindly donated by Arla Food Ingredients Group (Viby, Denmark) and is ≧
156 90% protein dry matter, 0.2% lactose, 0.2% fat, 4.5% ash, 5.0% moisture.
157
158 2.2. OPN mucilage production
159 OPN leaves were collected (Lavras, MG, Brazil), placed in polyethylene bags
160 and stored at −18 oC (Metalfrio DA 420, SP, Brazil) until use. OPN extraction was
161 performed by gridding 1 kg of leaves with 2.5 L of boiling water using an industrial
162 blender (Metvisa LG10, SP, Brazil) for 10 min. The mixture was then heated to 75 °C
163 using a thermostatic bath (Quimis q-215-2, SP, Brazil) for 6 hr. The blend was vacuum
164 filtered (Primar MC 1284, SP, Brazil) using three layers of organza fabric. The fiber
165 was removed by centrifugation of the filtrate (SP Labor, SP-701, SP, Brazil) (7 min,
166 4677 × g). The supernatant was diluted to make 1:3 (extract: ethanol, 95% purity); after
167 the precipitate was collected, frozen at −75 °C (Coldlab, CL120-86V, SP, Brazil), and
168 freeze-dried (−40 °C; 0.998 mbar; 5 days) (Edwards High Vacuum, L4KR, SP, Brazil).
169 The powder (6.08 ± 0.12% moisture; dry basis: 78.93 ± 0.29% carbohydrate, 8.89 ±
170 0.17% proteins, 9.99 ± 0.03% ash, 1.68 ± 0.16% lipids, 0.49 ± 0.29% fiber) was
171 hermetically packed and stored at 25 °C in vacuum desiccators until further use.
172
173 2.3. Titration and turbidity of WPI and OPN dispersions
174 Titratable acidity (MPT-2 autotitrator (Malvern Instruments, Worcestershire,
175 UK)) for WPI (5.0% w/w) and OPN (0.1% w/w) used NaOH (1.0 mol·L−1) and HCl
176 (0.1 and 1.0 mol·L−1) as titrants. Zeta potentials between pH 2.5 and 7.5, at 0.5
177 intervals, evaluated the overall surface charge of the biopolymers. Optical turbidity
7
178 evaluated the formation of soluble and insoluble complexes to determine the optimum
179 continuous phase pH for the emulsification step. 0.075/0.037% w/w WPI/OPN
180 dispersion turbidity was measured between pH 2.5 and 7.5 using UV-spectroscopy
181 (Ultrospec 3100 pro, Biochrom Ltd., Cambridge, UK) at λ = 600 nm (Doost et al.,
182 2019). Concentration of 0.075% w/w for WPI and 0.037% w/w for OPN were used to
183 avoid absorbance values > 1.0 (a.u.). Before titration, stock solutions were produced by
184 hydrating polymers overnight. The pH of all treatments was adjusted manually using
185 HCl 1 mol·L−1 or NaOH 1 mol·L−1.
186
187 2.4. Emulsion preparation
188 Total solids concentration (wall material + oil) for emulsions was 20.0% w/w.
189 The continuous phase combined WPI (11.5%, 11.0%, 10.5% w/w) and OPN mucilage
190 (0.5%, 1.0%, 1.5% w/w). OPN concertation limits were selected according to previous
191 findings (Lago et al., 2019). OPN mucilage was first dispersed in distilled water, heated
192 to 80 °C and constantly agitated for 1 hr. WPI, at various concentrations (Table 1), was
193 added to the OPN dispersion and stirred for 2 hr. Samples were hydrated overnight at 4
194 °C. The pH of the continuous phase was adjusted to 7.0 ± 0.1. The dispersed lipid phase
195 (8.0% w/w) was then prepared by mixing α-tocopherol (10.0% w/w of the lipid fraction)
196 in either CA or CO and mixed using a magnetic stir bar (Fisher Scientific, ON, CA) for
197 30 min at 25 °C. The two phases were combined and pre-homogenized (Caframo 2002,
198 Caframo Lab Solutions, ON, CA) at 1500 rpm for 5 min, at 25 °C. The pre-
199 homogenized samples were then passed through a high-pressure homogenizer
200 (EmulsiFlex-C5, Avestin, ON, CA), at 50 MPa, for 10 passes at 25 °C.
201
202 2.5. Rheological assessment of emulsions
203 The flow profile for emulsions was obtained using the coaxial cylinder
204 measurement system (CC27 12028, D = 13.331 mm; gap = 1.290 mm) coupled with a
205 Physica MCR 301 rheometer (Anton Paar, Ostfildern, Germany) and thermostatic bath
206 (Julabo F25, Julabo West Inc., PA, USA). Flow curves between 0.001 to 200 s−1 were
207 obtained from three continuous 2 min shear rate ramps (upward, downward and
208 upward) at 25 °C. To obtain fluid viscosity, both Newton's (Eq. 1) and Power Law (Eq.
209 2) models were fitted to the second rising curve, representing steady-state flow (Steffe,
210 1996):
211
8
τ = µγɺ (1)
τ = Kγɺ n (2)
212
213 where is shear stress (Pa), is Newtonian viscosity (Pa·s), is shear rate (s−1), is
214 the consistency index (Pa·sn), and is the flow behavior index (dimensionless). The
215 apparent viscosity at shear rate of 100 s−1 was reported. The rheological model was
216 fitted to a non-linear regression fit using the Statistical Analysis System 9.1.2 software
217 (SAS Institute Inc., Cary, U.S.A., 2008).
218
219 2.6. Average droplet diameter, polydispersity index and zeta potential
220 The polydispersity index (PDI) and average droplet diameter (di) of emulsions
221 were obtained using a 1:250 (sample: deionized water) dilution factor to avoid multiple
222 scattering effects using dynamic light scattering (Nanotrac Flex, Microtrac, PA, USA).
223 The refractive indices of canola oil, coconut oil, and the aqueous phase were 1.47, 1.45
224 and 1.33, respectively. Zeta potential (Zetasizer Nano ZS, Malvern Instruments,
225 Worcestershire, UK) was calculated using the Smoluchowski approximation from
226 electrophoretic mobility (Delgado, González-Caballero, Hunter, Koopal, & Lyklema,
227 2007; Ravindran, Williams, Ward, & Gillies, 2018).
228
229 2.7. Microencapsulation of α-tocopherol by freeze-drying
230 Microparticles containing α-tocopherol were made in triplicate and obtained by
231 freeze-drying the emulsions (Table 1). After freezing emulsions at −80 °C for 24 hr
232 (Forma 900 series, Thermo Scientific, OH, USA) they were lyophilized (Genesis 35EL,
233 Virtis, CA, USA) at 2.67 mbar, −40 °C, for 48 hr. The material was triturated using a
234 porcelain mortar and pestle and the powder was stored in dark sealed containers. The
235 drying yield (% w/w) was calculated as the powder content (g) obtained after freeze-
236 drying divided by total solids (g) in the emulsion (Pellicer et al., 2019).
237
238 2.8. Powder analysis
239 2.8.1. Moisture content, water activity (aw) and bulked density
240 The moisture content (%) was determined by gravimetric analysis (Gravity
241 convection oven Precision 18, GCA Corporation, IL, USA) at 105 °C, until a constant
242 weight was achieved (method 943.03, AOAC, 2012). Water activity was measured
243 using an Aqualab Vapor Sorption Analyzer (Meter Group, WA, USA) at 25 °C. The
9
244 bulk density ( , g·cm−3) was calculated by measuring the volume (

, cm3) of 1.0 g
245 (, g) of non-compact powder placed in a 10 mL graduated cylinder, as described by
246 Eq. 3 (Reboul, 2017; Sharif et al., 2017):
247
m
BV = (3)
V
248 2.8.2. Encapsulation efficiency and oil retention

249 The encapsulation efficiency and oil retention were performed as previously
250 described (Silva, Zabot, & Meireles, 2015). Briefly, the surface oil content was
251 determined by adding 15 mL hexane to 1.5 g of microparticles and vortexed for 2 min at
252 25 °C. The suspension was filtered using Whatman N° 1 filter paper (Ge Healthcare,
253 Shanghai, China). The retentate was washed 3 times in 20 mL of hexane. Residual
254 solvent was evaporated from the filter paper using gravity convection drying (Precision
255 18, GCA Corporation, IL, USA) at 60 °C until a constant weight was achieved. The
256 non-encapsulated oil ( , g) was determined by difference from the weight prior to and
257 following hexane extraction. Total oil ( , g) was measured by Soxhlet (Glas-Col RX,
258 IN, USA) using the AOAC official method 991.36 (AOAC, 2012). Encapsulation
259 efficiency (, % w/w) was calculated from Eq. 4:
260
 TO − SO 
EE (%) =   × 100 (4)
 TO 
261
262 Oil retention ( , %) was calculated from the ratio between total oil ( , g) and
263 initial oil weight used during emulsion preparation.
264
265 2.8.3. Scanning electron microscopy (SEM)
266 A small portion of each sample was placed on the SEM stubs coated with carbon
267 (600 Ultra Fine Norton SandWetTM, Worcester, MA, USA). Samples were then placed
268 in a vacuum sputter coater (Denton Vacuum Desk V, NJ, USA) and let equilibrate at a
269 vacuum pressure of 9 x 10-5 KPa before sputter coating gold-palladium blend (20 nm
270 thickness) onto the sample surface using a 20 mA deposition current. Powder
271 morphology was imaged using FEI Quanta 250 FEG-SEM (Thermo Fisher Scientific,
272 Oregon, USA) equipped with Schottky field emission gun and Everhart-Thornley
10
273 detector for secondary electrons. Imaging was controlled by the xT Microscope Control
274 software. An accelerating voltage of 10 kV was maintained throughout the microscopy.
275
276 2.8.4. Accelerated storage stability test
277 α-Tocopherol stability and bioaccessibility (section 2.9.) were assessed for CA
278 WPI:OPN (23:1) and CO WPI:OPN (23:1). An accelerated storage stability test
279 evaluated the chemical stability of α-tocopherol encapsulated microparticles (do Carmo
280 et al., 2018). ~ 25 g of powder, in Ziploc bags (165 mm x 149 mm), were stored at 60 ±
281 1.0 °C (Isotemp 60 L Incubator Gravity, Thermo Fisher Scientific, Langenselbold,
282 Germany) in either 21% relative humidity (RH) (saturated solution of NaOH) or 55%
283 (saturated solution of NaCl). Encapsulation efficiency (as described in section 2.8.2.)
284 were tested once a week for 5 weeks using Fourier-Transform Infrared Spectroscopy
285 (FT-IR) (section 2.8.6.) and α-tocopherol concentration was quantified using HPLC.
286 Microparticle α-tocopherol concentration was determined as previously reported
287 by Hategekimana, Masamba, Ma, & Zhong (2015). Extracting α-tocopherol from the
288 microparticle core was accomplished by dispersing 10 mg of powder in 5 mL of
289 methanol/acetonitrile (97:3 v/v). The mixture was sonicated for three-20 min cycles.
290 The dispersion was centrifuged (Sorvall LYNX 4000, Thermo Scientific,
291 Langenselbold, Germany) at 1000 x g for 20 min and the supernatant was collected. α-
292 tocopherol concentration was quantified using HPLC (Agilent 1100 Series, Agilent
293 Technologies, Waldbronn, Germany) equipped with a diode array detector (DAD)
294 (G1315B DAD detector, Agilent Technologies, Waldbronn, Germany) at 295 nm. A 20
295 µL sample was injected onto a C18 column (Agilent ZORBAX Eclipse Plus C18 Rapid
296 Resolution 4.6 x 150 mm 3.5 µm and Eclipse Plus C18 guard column 4.6 x 12.5 mm 5
297 µm) maintained at 40 °C. The mobile phase (93:3 v:v methanol: acetonitrile) had an
298 isocratic 1 mL·min−1 flow rate. Pure α-tocopherol, 10 mg· L−1 to 500 mg·L−1, was used
299 to generate a standard curve and fitted with a linear regression (y = 1.1403·x-1.0299, R2
300 = 0.996).
301 α-Tocopherol retention ( , dimensionless) was defined as the ratio between
302 α-tocopherol concentration as a function of time ( , mg·L−1) and its concentration
303 immediately after powder production ( , mg·L−1). Degradation of α-tocopherol follows
304 first-order kinetics (Syamila, Gedi, Briars, Ayed, & Gray, 2019), thus the half-life (/ )
305 was determined according to Eq. 5 and 6 (do Carmo et al., 2018):
11
C 
kt = − ln  t 
(5)
C 
 0
ln 2
t = (6)
1/2 k
306
307 where (days−1) is a first-order rate constant, (days) is the storage period, and /
308 (days) is the time required to degrade 50% of the α-tocopherol.
309
310 2.8.5. Sorption isotherms
311 Sorption and desorption isotherms of 700 mg of microparticles were obtained at
312 25 ± 0.1 ºC using an AquaLab Vapor Sorption Analyzer (VSA) (Decagon Devices, Inc.
313 Pullman, WA, USA) in the dynamic vapor sorption (DVS) mode. Water activity ranged
314 between 0.10 and 0.90 at 0.05 increments. Equilibrium was achieved when the rate of
315 change in mass as a function of time (dm/dt) was < 0.05 over two consecutive
316 measurements. To describe the sorption isotherm of α-tocopherol microparticles, the
317 GAB (Guggenheim, Ander- son and de Boer) model (Eq. 7) (Timmermann, Chirife, &
318 Iglesias, 2001) was fitted using a non-linear square fit (Statistica software version 8.0,
319 (Statsoft, Inc., Tulsa, USA)). According to literature, this model accurately quantifies
320 water sorption isotherms of foods (Lewicki, 1997) and must include a minimum of five
321 water activities (Aykın-Dinçer & Erbaş, 2018).
322
m Ckaw
m = 0
(7)
(1 − ka w )(1 − kaw + Cka w )
323
324 where is the equilibrium moisture content (g water/100 g dry basis); is the
325 monolayer moisture content (g water/100 g dry basis); is the water activity
326 (dimensionless); and are model constants (dimensionless). The coefficient of
327 determination (R2) and the mean relative percentage deviation modulus (<10%) was
328 used to evaluate the quality of the adjustments (Arslan & Togˇrul, 2005).
329
330 2.8.6. Fourier transform infrared (FTIR) spectroscopy
331 The FTIR spectroscopy, equipped with an attenuated total reflection (ATR) cell
332 (Pike Technologies, Madison, USA), assessed chemical degradation of α-tocopherol in
333 WPI/OPN microparticles (IRPrestige21, Shimadzu Corporation, Japan). Samples were
12
334 placed on the ATR cell and scanned 40 times at a resolution of 4 cm−1 in the mid-IR
335 region (600 to 4000 cm−1).
336
337 2.9. Bioaccessibility - TIM-1 simulated digestion
338 The TNO intestinal model (TIM-1) is a dynamic, robotic simulate
339 gastrointestinal system that mimics the stomach and upper intestinal compartments (i.e.,
340 duodenum, jejunum, and ileum), facilitating bioaccessibility studies under fed or fasted
341 conditions (Minekus et al., 1995; Ribnicky et al., 2014; Samtlebe et al., 2018;
342 Thilakarathna et al., 2016). This model has been widely validated and statistically
343 correlates to in vivo human studies (Anson et al., 2011; Déat et al., 2009; Eklund-
344 Jonsson, Sandberg, Hulthen, & Alminger, 2008; Larsson, Minekus, & Havenaar, 1997;
345 Marteau, Minekus, Havenaar, & Huis In’t Veld, 1997; Souliman, Blanquet, Beyssac, &
346 Cardot, 2006; Van De Wiele et al., 2007; Verwei, Freidig, Havenaar, & Groten, 2006).
347 The fed state protocol for the TIM-1 was used to assess the release profile of α-
348 tocopherol from microparticles over a 6 hrs. To mimic human physiological conditions,
349 these compartments were filled with preset solutions following a programmed protocol
350 and included: hydrochloric acid (1.00 mol·L-1 HCl), sodium bicarbonate (1.00 mol·L−1
351 NaHCO3), gastric solution (0.40% w/v HPMC and 0.04% w/v bile salts powder), small
352 intestinal electrolyte solution (SIES, 5.00 g·L−1 NaCl, 0.60 g·L−1 KCl, and 0.25 g·L−1
353 CaCl2), gastric enzyme solution (4.80 g·L−1 NaCl, 2.20 g·L−1 KCl, 0.22 g·L−1 CaCl2, 20
354 U·mL−1 lipase, 4800 U·mL−1 pepsin and 47 U·mL−1 amylase), 7.00% w/v pancreatin
355 solution and fresh porcine bile previously collect from a slaughterhouse (Conestoga
356 Meats, Breslau, CA). TIM-1 compartments were maintained at 37 ± 1 ºC during the
357 simulated digestion and pH was dynamically controlled (Table 2). The rate of gastric
358 emptying was preset with a half-time of 80 min (TNO, Zeist, The Netherlands).
359 The TIM-1 compartments were prefilled with starting residues to mimic in vivo
360 fed-state gastrointestinal conditions. The gastric starting residue was composed of 5.0 g
361 gastric enzyme solution and 5.0 g of gastric solution; the duodenal starting solution
362 consisted of 15.0 g of SIES, 15.0 g of 7.0% w/v pancreatin, 30.0 g of fresh bile and 1
363 mL trypsin solution (2 mg·mL SIES−1); the jejunal starting residue was 35.0 g SIES,
364 35.0 g 7.0% w/v pancreatin, and 70.0 g fresh bile; and the ileal starting residue
365 consisted of 140.0 g SIES. The solution used to simulate the ileal fluid was SIES and
366 the jejunal secretion was composed of 10% fresh porcine bile in SIES. Semi-permeable
367 filters (0.05 µm capillary membranes, Spectrum Milikros modules M80S-300-01P,
13
368 Repligen, Waltham, USA) were attached to the ileal and jejunal compartments in order
369 to obtain the micellar fractions representing the bioaccessible α-tocopherol fraction.
370 The fed ‘meal’ contained 10.0 g α-tocopherol WPI/OPN microparticles, 130.0 g
371 of gastric electrolyte solution (4.80 g·L−1 NaCl, 2.20 g·L−1 KCl, 0.22 g·L−1 CaCl2),
372 100.0 g water and 11.0 mg of amylase. The meal was added to the gastric compartment
373 containing 10.0 g of gastric starting residue. An additional 50.0 g of water was used to
374 rise the meal container and was added to the gastric compartment thus the total meal
375 was 300 g. Filtrates containing the bioaccessible α-tocopherol were analyzed by HPLC
376 (as described in the section 2.8.4) at 30, 60, 90, 120, 180, 240, and 300 min from the
377 jejunal and ileal compartments and the ileal efflux.
378 Cumulative α-tocopherol bioaccessibility (sum of jejunum + ileum filtrates) as a
379 function of time was fitted to the shifted-logistical model (Eq. 8) using a non-linear
380 analysis (Graphpad Prism 6.0 (La Jolla, CA)) (AlHasawi et al., 2018; Speranza et al.,
381 2013):
C asymp Casymp
C (t ) = − (8)
1 + e k (t c − t ) 1 + e kt c
382
383 where (%) is the % α-tocopherol released as a function of time (, min); !"#
384 (%) is the maximum α-tocopherol bioaccessibility; (min-1) is a rate constant; and $
385 (min) is the induction time, representing the time to release 50% of the total
386 bioaccessible α-tocopherol content. This model satisfies the boundary condition that at
387 =0 min, the bioaccessible α-tocopherol content is zero. Statistical significance between
388 formulations for fitted bioaccessibility parameters was evaluated. All analyses were
389 done in triplicate and expressed as mean ± standard deviation.
390
391 2.10. Statistical analysis
392 Analysis of Variance (ANOVA) determined statically significant differences
393 (p < 0.05) between treatments for both the emulsions and powders. Tukey post-hoc or
394 Student t-tests (p < 0.05) evaluated means and standard error.
395
396 3. Results and Discussion
397 3.1. Optimization of solution and emulsion characteristics required for microparticle
398 production
14
399 OPN and WPI were similarly charged (Fig. S.1A & B) and had minimum
400 turbidity, corresponding to low OPN-WPI interactions, when combined (Fig. S.1C) at
401 pH of 7; thus, it was selected as the optimal pH for further formulation. ζ-Potential of
402 OPN was negative across all pH values (2.5 < pH < 7.5) with an extrapolated pI <
403 pH=2.5 (Fig. S.1B), and the pI for WPI was 5.30 (Fig. S.1A).
404 Flow behavior of emulsions, prior to freeze drying, was dependent on both
405 biopolymer concentration and ratio as well as carrier oil (CO or CA). WPI:OPN (23:1)
406 in both CO and CA showed Newtonian behavior and fitted to the Power law (p < 0.05
407 and R2 > 0.998) (Table 3). Higher OPN concentrations (i.e., > 0.5% w/w (WPI:OPN
408 11:1 and 7:1)) were fitted to the Power law (p < 0.05 and R2 > 0.997) (Table 3). The
409 consistency index () and the apparent viscosity (η100) significantly increased (p < 0.05)
410 at higher OPN concentrations. and η100 were highest for CA WPI:OPN (7:1), which
411 contained 1.5% w/w OPN and CA, and agrees with previous reports (Junqueira et al.,
412 2018; Lago et al., 2019). With total solids constant, higher OPN concentrations had
413 higher viscosities attributed to the branched structure of OPN mucilage which contains
414 arabinogalactan chains (carbohydrate fraction) attached to proteins (Lago et al., 2019).
415 In all cases, CA presented statistically higher (p < 0.05) μ, and η100 values compared
416 to those prepared with CO due to the higher viscosity of pure CA (μ = 0.061 Pa·s)
417 compared to CO (μ = 0.050 Pa·s) when comparing treatments with similar
418 encapsulating composition (Table 3). The pseudoplastic behavior, fit to the Power law,
419 for both CO and CA presented higher apparent viscosity, values, and lower values
420 (p < 0.05) for treatments containing 1.5% w/w OPN. Increased pseudoplasticity was
421 observed with increasing OPN concentration (Table 3) which is consistent with similar
422 studies (Anvari & Melito, 2017; López-Castejón, Bengoechea, Espinosa, & Carrera,
423 2019).
424 CA WPI:OPN (7:1) emulsions had a bimodal distribution of larger particle sizes
425 as apparent by the elevated polydispersity index (PDI) (Table 4); and as such this wall
426 material formulation was not considered for further bioaccessibility studies. At 1.5%
427 w/w CA and OPN had larger droplets because of the high solution viscosity (Table 3).
428 Higher emulsion viscosity hinders oil-droplet disruption during homogenized, requiring
429 higher energy input to achieve a monomodal distribution leading to significantly higher
430 operating costs. Instability is also introduced with high biopolymer ratios and viscosities
431 including: 1) restricted diffusion and interfacial adsorption of surface-active molecules,
432 2) associative-electrostatic interactions bridge binding leading to flocculation; 3)
15
433 thermodynamic incompatibility between biopolymers leading to depletion flocculation;

434 and 4) etc. (Akbas, Soyler, & Oztop, 2018; Dickinson, 2019; Juttulapa, Piriyaprasarth,
435 Takeuchi, & Sriamornsak, 2017; Lago et al., 2019; McClements, 2015).
436 All formulations were controlled at pH 7, above the pI for both WPI and OPN
437 (Fig. S.1A & B), and had negative ζ-potentials (Table 4). Increasing OPN concentration
438 coincided with significantly (p < 0.05) decreased ζ-potential, arising from counter-ions
439 present in OPN mucilage which potentially shield electrostatic repulsion (as Na+, K+,
440 Mg2+, Ca+2, Mn2+) (Martin et al., 2017; McClements, 2015). Colloidal systems have
441 considerable kinetic stability when ζ-potential is greater than +30 mV or less than −30
442 mV (Shanmugam & Ashokkumar, 2014). Thus, it is expected that treatments CA
443 WPI:OPN (23:1) and CO WPI:OPN (23:1) would have higher stability considering also
444 their smaller droplet size diameter (Table 4). An increase in ζ-potential magnitude for
445 emulsions (Table 4) compared to biopolymers solutions (Fig. S.1A & B) may be
446 attributed to exposure of charged sites during homogenization that, in the native
447 conformation, were shielded (Saricaoglu, Gul, Besir, & Atalar, 2018; Tabilo-Munizaga
448 et al., 2019).
449
450 3.2. Microparticle characterization
451 Following microparticle production drying yield, moisture content, water
452 activity (aw), bulk density (BD), α-tocopherol encapsulation efficiency (EE) and oil
453 retention (Table 5) were assessed to find the optimal formulation for assessing long-
454 term stability and α-tocopherol bioaccessibility. Although drying yield varied between
455 formulations, moisture content nor aw statistically differed between treatments (p >
456 0.05) (Table 5). More importantly, aw ranged between 0.198 ± 0.002 to 0.205 ± 0.006
457 which was desirable as it is sufficiently low to prevent degrative reactions (Caliskan &
458 Dirim, 2016), microbial growth, and caking (Goyal et al., 2015); while above the
459 optimal range for lipid oxidation in powders (0.1 < aw < 0.2) (Frankel, 2012). The bulk
460 density ( ) was between 0.15 ± 0.00 and 0.22 ± 0.01 g·cm-3 irrespective of the oil
461 used, and formulations with low OPN concentrations had higher arising from
462 viscosities (Table 3) and smaller oil droplet diameter (Table 4) in precursor emulsions.
463 Upon freeze drying, the consequence is smaller pores through the dried structure
464 resulting in finer powder when triturated (Aghbashlo, Mobli, Rafiee, & Madadlou,
465 2012). Irregularly sized and shaped microparticles result in external voids, hindering
16
466 particles compaction and lower (Rajam & Anandharamakrishnan, 2015).

467 Encapsulation efficiency () reflects protection conferred to α-tocopherol against
468 environmental conditions (Fioramonti et al., 2017). Formulations containing CO
469 exhibited higher % than those prepared with CA (Table 5), regardless of the
470 WPI/OPN ratio since CO is solid and CA is liquid at room temperature. For CA, there
471 was a positive correlation between % and emulsion droplet size, which has been
472 shown to minimize oil migration and amount of unencapsulated oil (Fioramonti et al.,
473 2017; González, Martínez, Paredes, León, & Ribotta, 2016). Oil retention, described as
474 mass of oil in the particle divided by precursor emulsion total solids, did not
475 significantly differ between samples (Table 5), which others have reported (Silva,
476 Azevedo, Cunha, Hubinger, & Meireles, 2016; Silva, Zabot, Cazarin, Maróstica, &
477 Meireles, 2016).
478 Higher OPN concentrations led to darker powders, as illustrated moving left to
479 right across Fig. S2, which is expected since the OPN mucilage powder is dark
480 (Oliveira et al., 2019). It is also apparent that rougher powder texture (Fig. 1) coincides
481 with more viscous emulsions (Table 3), which is attributed to harder structures
482 produced after freeze-drying and thus larger particles when titrated. Agglomerates in
483 CA WPI:OPN (7:1) and its low % (67.26 ± 0.21% w/w), suggests that free oil
484 migrating from the core to surface is responsible for particle aggregation (Fioramonti et
485 al., 2017). Porous, irregular continuous surfaces, similar to glass-like flakes previously
486 reported for freeze-dried powders are observed herein (Fig. 1) (Chranioti, Chanioti, &
487 Tzia, 2016; Fioramonti et al., 2017; Pellicer et al., 2019; Vardanega, Muzio, Silva,
488 Prata, & Meireles, 2019). The undulating surface morphologies (Fig. 1C and 1F), reflect
489 lower EE% with high OPN concentration (Table 5); while Fig. 1A and 1D have
490 smoother surface morphology.
491
492
493
494
17
495
496
497
498
499
500 Figure 1. SEM images of freeze-dried α-tocopherol microparticles produced from WPI/OPN
501 and CA or CO-in-water emulsions: CA WPI:OPN (23:1) (A); CA WPI:OPN (11:1) (B); CA
502 WPI:OPN (7:1) (C); CO WPI:OPN (23:1) (D);CO WPI:OPN (11:1) (E); CO WPI:OPN (7:1)
503 (F). Scale bars are provided on each micrograph.
504
505 3.3. Long-term microparticle stability
506 Based on the emulsion characteristics (e.g. low viscosity and monomodal
507 particle size distribution) and physical properties (e.g., low moisture content and water
508 activity, and higher drying yield, % and ) of the microparticles, CA WPI:OPN
509 (23:1) and CO WPI:OPN (23:1) were selected to test storage stability and α-tocopherol
510 bioaccessibility. Microparticles entrained the oil phase as there was no significant
511 difference in EE% and α-tocopherol retention indicated (p > 0.05) (Fig. 2A and 2B)
512 over a 5-week accelerated self-stability test at 60 ºC and 21% (saturated NaOH solution)
513 or 55% (saturated NaCl solution)) relative humidity. Both relative humidities are below
514 the critical point (i.e., RH = 65%) where water absorption occurs at significantly faster
515 rates (Fig. 2C & D). Low initial moisture content of microparticles translates to
516 improved stability (e.g., reduced rates of: microbial growth, enzymatic and non-
517 enzymatic chemical reactions) contributing to extend the product shelf-life (Otálora,
18
518 Carriazo, Iturriaga, Nazareno, & Osorio, 2015). α-tocopherol retention, after 35 days,
519 was dependent on the lipid phase (Fig. 2B and Table 6). For CO containing
520 microparticles α-tocopherol retention ( was > 85% at both 21 and 55 % RH. In
521 contrast, CA microparticles had a significant decrease in , with values less than
522 30% of initial α-tocopherol microparticles concentration (Fig. 2B). CO is comprised
523 primarily of saturated fatty acids that are less susceptible to oxidation (de Moura e Dias
524 et al., 2018). Also, higher for CO microparticles may be associated with tighter
525 fatty acid chains packing resulting in more dense structures, which potentially mitigate
526 oxygen permeability (Otálora et al., 2015; Wang et al., 2017). Microparticle α-
527 tocopherol retention, as a fucntion of time, was fittecd to a first order equation (0.949 <
528 R2 < 0.983), to obtain the degradation rate constant, (days−1), and the time until half
529 the reaction has completed, / (days) (Table 6). Clearly, stability of α-tocopherol
530 depended on carrier oil; CO had lower values and higher / , compared to CA, which
531 was independent of RH. These results concur with findings, suggesting α-
532 tocopherol stability is improved when CO was the lipid carrier.
533 Conversely, CA microparticle α-tocopherol degradation kinetics are dependent
534 on RH (p < 0.05). Higher unsaturated fatty acids content of CA are prone oxidation,
535 which accelerates in the presence of water (Vergara, Saavedra, Sáenz, García, & Robert,
536 2014). CA containing microparticles stored at RH = 21% had greater stability (=0.04 ±
537 0.00 days−1 and / = 18.83 ± 0.56 days) compared to RH = 55% ( = 0.04 ± 0.00
538 days−1 and / = 15.65±0.47 days) implying that α-tocopherol is degraded to a greater
539 extent when CA is used as the lipid carrier compared to CO. The less dense lipid core
540 facilitates oxygen diffusion through the encapsulating matrix thereby accelerating α-
541 tocopherol degradation and shortening shelf-life (Hategekimana et al., 2015).
542 Irrespective of the carrier oil used, low water adsorption capacity was observed at low
543 aw, in contrast with a sharp increase in moisture content for aw > 0.650; thus, materials
544 have extended shelf-life when stored at RH < 65%. The GAB model accurately fit
545 isotherms for the treatment prepared with CA (R2 = 0.996 (adsorption curve) and 0.998
546 (desorption curve)) and for CO (R2 = 0.997 (adsorption curve) and 0.994 (desorption
547 curve)). The monolayer moisture contents, predicted by the model, were 4.62 g
548 H2O/100 g d.b. and 5.01 g H2O/100 g d.b. for the adsorption curves of microparticles
549 with CA or CO, respectively. For the desorption curves CA and CO were 5.51 g
550 H2O/100 g d.b. and 6.06 g H2O/100 g d.b., respectively. Similar monolayer moisture
19
551 contents in both CO and CA α-tocopherol microparticles are expected due to identical
552 encapsulation wall material composition. The monolayer moisture content, or water
553 strongly adsorbed to protein and carbohydrate fractions of the WPI and OPN, represents
554 the ideal water content required to produce chemically and microbiologically stable
555 powders (Alpizar-Reyes et al., 2018). GAB constants, , were 13.32 (adsorption
556 isotherm) and 17.21 (desorption isotherm) for the microparticles containing CA, in
557 comparison with 23.90 (adsorption isotherm) and 26.17 (desorption isotherm) for
558 treatments with CO. This parameter is a measure of the energy involved in the water
559 adsorption at the monolayer binding sites (de Sá Mendes et al., 2019; Téllez-Pérez,
560 Sobolik, Montejano-Gaitán, Abdulla, & Allaf, 2015). Higher values correlate to larger
561 differences in enthalpy between the monolayer and multilayer molecules (Quirijns, Van
562 Boxtel, Van Loon, & Van Straten, 2005). Higher values obtained for microparticles
563 containing CO suggest stronger water binding at the monolayer.
564 Finally, the GAB constant was calculated to be 0.92 (adsorption isotherm) and
565 0.88 (desorption isotherm) for the treatment prepared with CA, while for microparticles
566 containing CO it showed values equal to 0.92 (adsorption isotherm) and 0.87
567 (desorption isotherm). is a measure of the energy of interaction between water
568 molecules adsorbed at the monolayer and at a distant adsorption site (Alpizar-Reyes et
569 al., 2018). More structured molecules in the multilayers (layers adjacent to the
570 monolayer) have lower values, while values close to 1 implies the water molecules
571 not included in the monolayer behavior, as free bulk water (Quirijns et al., 2005).
572 values were close to 1 suggesting fewer interactions between water and the
573 encapsulating biopolymers. According to Lewicki (1997), isotherms are adequately
574 described when and values are between 0.24 < ≤ 1 and 5.65 ≤ ≤ ∞, as it
575 ensures the predicted values do not differ from the real capacity of the monolayer more
576 than ±15.5%. Besides, < 1 and > 2 are an indicative of Type-II sigmoid behavior
577 (Brunauer, Emmett, & Teller, 1938), which accounts for the existence of multilayers
578 arising due to colligative effects (i.e., water confined in capillaries) and surface water
579 interactions (de Sá Mendes et al., 2019; Erbaş, Aykın, Arslan, & Durak, 2016).
580
20
A B
C D
581 Figure 2. (A) Encapsulation efficiency (%) of α-tocopherol microparticles during storage,
582 according to the relative humidity (21%, dark bars; 55%, light bars) and lipid phase (●, canola
583
584
585
oil; \, coconut oil). (B) α-tocopherol retention throughout time for the treatments composed
with: canola oil and stored in a RH = 21% (●); canola oil and stored in a RH = 55% (△);
coconut oil and stored in a RH = 21% (■); coconut oil and stored in a RH = 55% ( ).
◇
586 Absorption (○) and desorption (□) isotherms and GAB model fit for the treatments: CA
587 WPI:OPN (23:1) (C), and CO WPI:OPN (23:1) (D).
588
589 WPI peaks, obtained with FTIR, at 1635 cm−1 (amide I) and 1525 cm−1 (amide
590 II) (Fig. 3A) are related to peptide bond vibrations (-CO-NH) (Andrade et al., 2019;
591 Tan, Ebrahimi, & Langrish, 2019; Tan, Zhong, & Langrish, 2019). Peaks at 1442, 1384,
592 1228 and 628 cm-1 correspond with bending of -CH2, streching of -COO-, stretching -C-
593 C vibrations and ring -C-H deformation (Esfanjani, Jafari, Assadpoor, & Mohammadi,
594 2015; Singh, Singh, Karthick, Tandon, & Prasad, 2018; Tan, Zhong, et al., 2019;
595 Zhang, Peng, Ma, & Zeng, 2019). Peaks at 3050 and 2947 cm−1 are associated with
596 aliphatic carbons -C-H(CH2) and -C-H(CH3) (Andrade et al., 2019; Botelho, Reis,
597 Oliveira, & Sena, 2015). The region between 1200-900 cm-1(stretching -C-C or -C-OH
598 vibrations) is characteristic of carbohydrates, such as lactose present in WPI (Andrade
599 et al., 2019; Darra et al., 2017). The broad peak between 3500-3100 cm−1 (stretching O-
600 H band) suggests the presence of water, carbohydrate -O-H hydrogen bonds (Monrroy,
601 García, Ríos, & García, 2017) or potentially stretching -N-H vibrations of amides
602 (Conceição et al., 2014; Mól et al., 2019). Similar peaks were found in the spectrum
603 obtained for OPN (Fig. 3B), which contains both proteins and carbohydrates, at 3360
604 cm−1 (stretching O-H vibrations), 2912 cm−1 (aliphatic carbons -C-H(CH2) and -C-
605 H(CH3)), 1627 cm−1 (amide I), 1377 cm−1 (-COO-), 1249 cm−1 (stretching -C-C
21
606 vibrations) and 669 cm−1 (-C-H bending deformation). Peaks at 1317 and 1033 cm−1 are
607 associated with amide III (stretching -C-N) from the oligopeptide covalent bonds (Tan,
608 Zhong, et al., 2019), and stretching -C-O vibration of alcohols (Andrade et al., 2019).
609 The peak at 806 cm−1 correspond with ring -C-H out-of-plane bending (Singh et al.,
610 2018). FTIR spectra of OPN mucillage corresponds to previous reports of Conceição et
611 al. (2014).
612 FTIR spectra of freeze-dried microparticles (Fig. 3C-F) maintained similar peaks
613 as observed to WPI (Fig. 3A) and OPN (Fig. 3B). α-tocopherol exhibited absorbance
614 bands at 3473 cm−1 (stretching -O-H), 2927 and 2868 cm−1 (asymmetric and symmetric
615 stretching vibrations of –CH2 and –CH3), respectively (Che Man, Ammawath, &
616 Mirghani, 2005). Absorbance at 1461 cm−1 coninsides with phenyl skeletal or methyl
617 asymmetric bending, while 1378 cm−1 corresponds to methyl symmetric bending, 1262
618 cm−1 for –CH2 stretching bending and 1086 cm−1 for plane bending of phenyl. These
619 peaks are clearly present in microencapsulated of α-tocopherol in freeze-dried powders
620 (Fig. 3C-F). The lipid phase (CA or CO) presented vibrations at ~ 3008 cm-1 atributed
621 to stretching of cis double bonds (-C=C-H), at 2920-2929 and 2850-2858 cm-1
622 representing asymmetrical and symmetrical -C-H stretching of aliphatic groups of
623 organic fatty acids, and at 1737-1749 and 1244-1236 cm−1 (stretching -C=O of
624 triglycerides or -C-O-C of esters, respectively). Peaks at 1460 cm−1 (bending vibrations
625 of -CH2 and -CH3 aliphatic groups), 1386-1396 cm-1 rocking vibrations of -CH bonds of
626 cis-disubstituted olefins in canola oil, at 1149-1155 cm−1 (stretching -C-O vibration of
627 aliphatic esters) and at 1072-1092 cm−1 (stretching -CO band of ether linkages or
628 stretching -COH of alcohols) (Beneš et al., 2017; Khorasani, Ataei, & Neisiany, 2017;
629 Mhaske, Condict, Dokouhaki, Katopo, & Kasapis, 2019; Ozulku, Yildirim, Toker,
630 Karasu, & Durak, 2017; Talpur et al., 2015). FTIR spectra of samples with CO
631 remained constant over 35 days of storage (Fig. 3E and 3F) indicatning α-tocopherol did
632 not undergo degradation or oxidation, as previously predicted by the results and α-
633 tocopherol retention (Fig. 2B). In contrast, samples with CA, observed peaks at forming
634 in time at 1155, 854 and 954 cm−1, which were atributed to the formation of aromatic -
635 CH bending vibrations and -C-O-C stretching epoxides (Khundamri, Aouf, Fulcrand,
636 Dubreucq, & Tanrattanakul, 2019), indicating α-tocopherol is being converted to
637 quinones via epoxide formation (Hategekimana et al., 2015).
22
A 1033
B 1635
1525
Absorbance (a.u.)
Absorbance (a.u.)
1442 678
669 1228
1627
1384 1141
OPN
806 WPI
929
1317
1249
3360 1377 3275
3055 2947
2912
4000 3500 3000 2000 1500 1000 500 4000 3500 3000 2000 1500 1000 500
-1 -1
638 Wavenumber (cm ) Wavenumber (cm )
639
1155
1072
1633 1527
C 1460 1244 678
D 1635 1533
1460
1232
673
1087
1155 2928 1386
1396 854 0 days
3286 2929 0 days 3282 2858 1739
3055 2854 3055
1745
7 days
Absorbance (a.u.)
7 days
Absorbance (a.u.)
954
14 days
14 days
24 days 24 days
28 days 28 days
35 days
35 days
4000 3500 3000 2000 1500 1000 500

4000 3500 3000 2000 1500 1000 500
-1 -1
640 Wavenumber (cm ) Wavenumber (cm )
641
1631 1149
E 1527
1155 F 1639 1531 1236
1446
1440 1244 678 1097 677
1390 1072 1392
0 days 3286 2920 0 days
3286 2922 2854 1737
2850 7 days
1749
Absorbance (a.u.)
7 days
Absorbance (a.u.)
14 days
14 days
24 days
24 days
28 days
28 days
35 days
35 days
4000 3500 3000 2000 1500 1000 500 4000 3500 3000 2000 1500 1000 500
-1 -1
Wavenumber (cm )
642 Wavenumber (cm )
643 Figure 3. FTIR spectra of WPI (A); OPN (B); and freeze-dried α-tocopherol microparticles
644 stored at low (21%, C and E) or high (55%, D and F) relative humidity, according to the oil
645 carrier used (canola oil, C and D; coconut oil, E and F).
646
647 3.4. TIM-1 simulated digestion
648 Microencapsulated powders of CA WPI:OPN (23:1) or CO WPI:OPN (23:1)
649 and liquid formulations (CA and CA WPI:OPN (23:1) emulsions) each contained 2.5%
650 w/w α-tocopherol was assessed using the TIM-1 simulated gastrointestinal tract (Fig. 4).
651 Bioaccessiblilty herein represents the α-tocopherol released from the microencapsulates
652 into the luminal fluid and is available for absorbtion (AlHasawi et al., 2018).
653 Cumulative bioaccessbility followed sigmoidal trends, with a characteristic initial time
654 period of ~ 60 min for microencapsulates and was higher for CA compared to CO (Fig.
23
655 4 jejunum + ileum). A non-linear shifted-logistical model was fitted (R2 > 0.9828) to the
656 sigmodial bioaccessibility curves to obtain the bioaccessible fraction (%), rate constant
657 (min−1) and induction time (min) (or a time to achieve half of the bioaccessible fraction)
658 (Fig. 4 bottom row). The fitted total bioaccessibility from microencapsulates was 42.42
659 ± 1.78% for CO, and 54.98 ± 1.89% for CA. The α-tocopherol loaded CA OPN/WPI
660 emulsion had a significnatly lower bioaccessibility (i.e., 39.6 ± 0.90%), compared to the
661 microencapulated powder, demonstrating the utitlity of OPN/WPI encapsulates at
662 improving bioactive stability and bioavailabity. Bioaccessibility for the control (α-
663 tocopherol + CA) was lower than all other formulations (9.48 ± 0.44%), illustrating the
664 utility of hydrocolloids in emulsions and microparticles to enhance encapsulating of α-
665 tocopherol microencapsulation.
666 The bioaccessible α-tocopherol fraction was greater for CA WPI:OPN (23:1)
667 compared to CO WPI:OPN (23:1) microparticles. The long-chain fatty acids, higher in
668 CA, are more hydrophobic with larger solubilization capacities for lipophilic substances
669 compared to medium-chain fatty acids, which has previously been postulated to increase
670 bioaccessibility (Verkempinck et al., 2018). Others have suggested that the 14 carbon,
671 non-polar chain of α-tocopherol may ineffectivly pack in micelles comprised of
672 medium chain fatty acids (Yang & McClements, 2013). Higher bioactive
673 bioaccessibility in CA versus CO result is in aggrement β-carotene and α-tocopherol
674 bioaccessibility in treatments containing different chain length tryglycerides (Nagao,
675 Kotake-Nara, & Hase, 2013; Yang & McClements, 2013). The rate constant () as well
676 as the induction time ($ ) were statiscally similar (p > 0.05) for samples prepared with
677 CA or CO (Fig. 4). The and $ were equal to 0.021±0.002 min−1 and 150.7 ± 4.6 min
678 when using CA, in comparison with 0.020 ± 0.002 min−1 and 138.6 ± 5.2 min for
679 powders containing CO, respectively. For these parameters, the lipid carrier did not
680 affect the α-tocopherol release from the core of the microparticle to the digestive
681 medium. This result was attributed to the similar wall material composition for both
682 samples, wich were disrupted in the same rate and throughout similar range of time.
24
683
684
685
◇
Figure 4. Cumulative bioaccessible fractions of α-tocopherol (%) obtained from the Jejunum, Ileum and
combined, 2.5% w/w in all samples fed into the TIM, for CA ( ), CA WPI:OPN (23:1) (emulsion) (△),
686 CA WPI:OPN (23:1) (○), CO WPI:OPN (23:1) (□) (first line in fig.). Fitted parameters (Bioaccessible
687 fractions (%), Rate constant (min-1) and Induction time (min)) from the shifted-logistical adjusted model
688 to the cumulative bioaccessible fractions obtained from the Jejunum, Ileum and combined (second line in
689 fig.). Different letters indicate significance within groups. Fitted parameters that had no overlap between
690 their 95% confidence intervals were considered statistically different.
691
692
693 4. Conclusion
694 Microparticles comprised on WPI and OPN obtained by freeze-drying canola or
695 coconut oil-in-water emulsions effectively encapsulated α-tocopherol while maintaining
696 high drying yields, low moisture contents and water activities. Microparticles obtained
697 from emulsions with higher OPN concentration had higher apparent viscosity, which
698 translated to lower encapsulation efficiency and bulk density, and did not affect oil
699 retention. α-tocopherol retention and degradation kinetics were greater when CA was
700 the carrier compared to CO; this was attributed to higher unsaturated fatty acid content
701 of CA, which has faster rates of α-tocopherol oxidation. OPN/WPI microparticles
702 confer chemical and microbiological stability at RH < 65 % during accelerated shelf-life
703 testing after 5 weeks at 60oC Finally, total in vitro bioaccessibility of α-tocopherol,
704 assesssed using the TIM-1 simulated digestive model, was significantly higher for CA
705 due to the presence long-chain fatty acid chains which have been shown to improve α-
706 tocopherol bioaccessibiltiy. The fitted shifted-logistical parameters, rate constant and
707 induction time, were not influenced by carrier oil.
708
709 Declaration of interest
710 The authors declare there is no conflict of interest for this research.
25
711
712 Funding
713 This study was financially supported by the Coordenação de Aperfeiçoamento
714 de Pessoal de Nível Superior – Brasil (CAPES) - Finance Code 001; Conselho Nacional
715 de Desenvolvimento Científico e Tecnológico – Brasil (CNPq) (Grant numbers
716 478376/2013-8 and 308043/2015-4); and the Fundação de Amparo à Pesquisa do
717 Estado de Minas Gerais – Brasil (FAPEMIG) (Grant numbers CAG - APQ-01308-12
718 and CAG - APQ-03851-16). Funding in Canada was received from the Canadian
719 Foundation for Innovation, NSERC Discovery and Canada Research Chair programs.
720
721 Acknowledgments
722
723 The authors gratefully acknowledge support from funding agencies (CAPES,
724 CNPq, FAPEMIG) for the financial support and the laboratory infra-structure provided
725 by the Food Science Departments of the Federal University of Lavras (BR) and
726 University of Guelph (CA). M.A.R. also thankfully acknowledges support from
727 Canadian Foundation for Innovation, NSERC Discovery and Canada Research Chair
728 programs.
729
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1256
1257
1258
1259
42
1260 Table 1. Emulsions composition.

Treatment Oil type WPI (% w/w) OPN (% w/w) Identification
1 Canola 11.50 0.50 CA WPI:OPN (23:1)
2 Canola 11.00 1.00 CA WPI:OPN (11:1)
3 Canola 10.50 1.50 CA WPI:OPN (7:1)
4 Coconut 11.50 0.50 CO WPI:OPN (23:1)
1261 WPI: whey protein isolate; OPN: ora-pro-nobis mucilage; CA: canola oil; CO: coconut oil.
1262
1263
1264 Table 2. Preset pH values of the TIM-1 compartments.
Compartment Predetermined pH levels
Duodenal 5.5
Jejunal 6.5
Ileal 7.4
Stomach (0 min) 6.5
Stomach (30 min) 4.2
1265
1266
1267
1268 Table 3. Newton and Power law fitted rheological parameters of OPN /WPI/Oil-in-
1269 water emulsions.
Treatment Newton law
μ (Pa·s)* RMSE R2
WPI:OPN (23:1)
CA 0.009±0.000a 0.014 0.999
CO 0.006±0.000b 0.016 0.998
Power law
(Pa·sn) * (-)* RMSE R2 η100 (mPa·s)*
WPI:OPN (11:1)
CA 0.036±0.001b 0.898±0.004b 0.008 0.999 22.750±0.041b
CO 0.019±0.002a 0.952±0.020c 0.016 0.999 16.330±0.874a
WPI:OPN (7:1)
CA 0.076±0.004d 0.840±0.014a 0.100 0.997 38.667±2.509c
CO 0.053±0.002c 0.847±0.001a 0.024 0.999 26.183±1.007b
*
1270 Different letters in columns indicate significant statistical differences (p < 0.05) by Tukey test.
1271 WPI: whey protein isolate; OPN: ora-pro-nobis mucilage; CA: canola oil; CO: coconut oil; RMSE: root-
1272 mean-square error; μ: Newtonian viscosity; : consistency index; : flow behavior index; η100: apparent
1273 viscosity at 100 s−1.
1274
1275
1276
43
1277 Table 3. Average droplet diameter (di) and ζ-potential results for emulsions prepared according
1278 to the continuous phase composition.
Treatment di (nm)* Volume (%) PDI (-)* ζ-potential (mV)*
WPI:OPN (23:1)
CA 287.00±4.25a,b 100.00 0.203±0.033a,b −50.08±0.68a
CO 230.37±5.31a 100.00 0.144±0.070a −49.63±0.41a
WPI:OPN (11:1)
CA 389.67±10.27b,c 100.00 0.269±0.042a,b −44.21±0.61b
CO 318.00±2.40a,b,c 100.00 0.252±0.026a,b,c −45.12±0.28b
WPI:OPN (7:1)
CA 1608.33±177.45d 52.77±5.72 0.443±0.036c −40.03±0.20d
355.33±71.35b,c 47.23±5.72
CO 402.67±13.69c 100.00 0.364±0.082b,c −42.02±0.69c
*
1279 Different letters in the column indicate significant statistical differences (p < 0.05) by Tukey test. WPI:
1280 whey protein isolate; OPN: ora-pro-nobis mucilage; CA: canola oil; CO: coconut oil; PDI: polydispersity
1281 index.
1282
1283 Table 4. Physico-chemical parameters for the α-tocopherol microparticles produced by freeze-
1284 drying.
Yield Moisture
Treatment aw*
(%)* (%)* (g·cm-3)* (% w/w)* (%)*
WPI:OPN (23:1)
CA 87.06±1.71c 2.45±0.09a 0.199±0.001a 0.20±0.00b 74.12±0.41b 77.87±0.48a
CO 86.03±0.63c 2.47±0.10a 0.198±0.002a 0.22±0.01c 79.77±0.41d 77.88±1.87a
WPI:OPN (11:1)
CA 79.64±1.28b 2.46±0.10a 0.205±0.006a 0.18±0.00b 73.05±0.72b 79.93±1.09a
CO 82.91±0.60b,c 2.41±0.12a 0.201±0.001a 0.19±0.01b 79.31±0.48c,d 76.95±0.44a
WPI:OPN (7:1)
CA 74.43±0.31a 2.41±0.11a 0.203±0.005a 0.16±0.00a 67.26±0.21a 79.33±0.58a
CO 74.05±2.91a 2.42±0.05a 0.205±0.006a 0.15±0.00a 77.42±0.97c 79.85±1.97a
*
1285 Different letters in the column indicate significant statistical differences (p < 0.05) by Tukey test. WPI:
1286 whey protein isolate; OPN: ora-pro-nobis mucilage; CA: canola oil; CO: coconut oil; aw: water activity;
1287 : bulk density; : encapsulation efficiency; : oil retention.
1288
1289 Table 5. α-Tocopherol retention after 35 days of storage and degradation rate constants
1290 of microparticles produced with CA or CO oil stored in different relative humididy, at
1291 60 ºC.
Sample (-)*,** (day-1) *,** / (days) *,** R2
Canola oil (RH=21%) 0.290±0.013A 0.037±0.001A 18.835±0.559A 0.983
B B B
Canola oil (RH=55%) 0.234±0.017 0.044±0.001 15.649±0.469 0.958
Coconut oil (RH=21%) 0.890±0.014a 0.004±0.000a 179.804±18.740a 0.949
Coconut oil (RH=55%) 0.872±0.012a 0.005±0.000a 154.048±12.920a 0.958
*
1292 Different capital superscripts in columns indicate significant difference (p < 0.05) for CO samples by t
1293 test. **Different lowercase letters in the column indicate significant difference (p < 0.05) for samples with
1294 CO by t test. RH: relative humidity; : α-tocopherol retention after 35 of storage; : first order rate
1295 constant; / : half-life time.
Highlights
• Encapsulation of α-tocopherol in microparticles obtained from freeze-drying o/w

emulsions using natural polymers
• Microcapsules presented high yield and oil retention with low moisture and aw
• α-tocopherol bioaccessibility was altered by unsaturated/medium-chain fatty acids
• WPI/OPN powders are effective encapsulatation materials and would be an excellent

functional additive
The authors declare that:
1. The manuscript represents original scientific contribution, which has not been
previously published elsewhere and it is not under consideration for publication in
another journal;
2. All authors have approved the manuscript and agree with its submission to Food
Hydrocolloids;
3. The authors declare that they have no conflict of interest;
3. The authors have understood the copyright issues and agree to the terms and
agreement of the journal;
4. The corresponding author accepts responsibility for releasing this material on
behalf of any and all co-authors.

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Effect of carrier oil on α-TOCOPHEROL encapsulation in ora-pro-nobis (Pereskia

To appear in: Food Hydrocolloids

Received Date: 21 October 2019

© 2020 Published by Elsevier Ltd.

Isabelle Cristina Oliveira Neves: Project administration, Conceptualization, Methodology,

1 EFFECT OF CARRIER OIL ON α-TOCOPHEROL ENCAPSULATION IN

76 Microencapsulation forms a protective boundary layer around bioactives which

144 bicarbonate (≧ 99.7%), (hydroxypropyl)methylcellulose (HPMC, mol. Wt. ~86 kDa),

244 bulk density ( , g·cm−3) was calculated by measuring the volume (

248 2.8.2. Encapsulation efficiency and oil retention

433 thermodynamic incompatibility between biopolymers leading to depletion flocculation;

466 particles compaction and lower  (Rajam & Anandharamakrishnan, 2015).

4000 3500 3000 2000 1500 1000 500

744 M. G., … Rogers, M. A. (2017). In vitro measurements of luminal viscosity and

778 International, 102, 1–7.

846 Hydrocolloids, 87, 71–82.

880 stabilized oil-in-water emulsions. Food Hydrocolloids, 96, 209–223.

914 and Bioprocess Technology, 6(3), 628–647. https://doi.org/10.1007/s11947-012-

948 Kelly, P. (2019). Chapter 3 - Manufacture of Whey Protein Products: Concentrates,

1016 Cotter, I. López-Expósito, C. Kleiveland, T. Lea, A. Mackie, … H. Wichers (Eds.),

1118 Surfaces A: Physicochemical and Engineering Aspects, 529, 550–559.

1220 redispersion properties. Food Research International, 116, 932–941.

1254 Engineering, 245(August 2018), 149–156.

1260 Table 1. Emulsions composition.

• Encapsulation of α-tocopherol in microparticles obtained from freeze-drying o/w

• α-tocopherol bioaccessibility was altered by unsaturated/medium-chain fatty acids

• WPI/OPN powders are effective encapsulatation materials and would be an excellent

S-ar putea să vă placă și

244 bulk density ( , g·cm−3) was calculated by measuring the volume (

466 particles compaction and lower (Rajam & Anandharamakrishnan, 2015).